Biomedical tests are frequently based on detecting an interaction between a molecule, which is present in known quantity and position (i.e. the molecular probe) and an unknown molecule to be detected or unknown molecules to be detected (i.e. the molecular target molecules). In modern tests the probes are deposited in the form of a substance library on supports, the so-called micro-arrays or chips so that one sample can be analysed simultaneously in parallel on a plurality of probes (D. J. Lockhart, E. A. Winzeler, Genomics, gene expression and DNA arrays; Nature 2000, 405, 827-836). In order to fabricate the micro-arrays the probes are usually immobilised in a predetermined manner on a suitable matrix, described for example in WO 00/12575 (see for example U.S. Pat. No. 5,412,087, WO 98/36827) or are produced synthetically (see for example U.S. Pat. No. 5,143,854).
The interaction between the probe and the target molecule is usually detected as follows: after fixing the probe or the probes in a predetermined manner on a specific matrix in the form of a micro-array, the targets are brought in contact with the probes in a solution and incubated under defined conditions. As a result of the incubation, a specific interaction takes place between probe and target. The binding observed in this case is definitely more stable than the binding of target molecules to probes which are not specific for the target molecule. In order to remove target molecules which have not been specifically bound, the system is washed with suitable solutions or heated.
The specific interaction between a target and its probe can then be detected by a plurality of methods which generally depend on the type of marker which has been incorporated into target molecules before, during or after the interaction of the target molecule with the micro-array. Such markers typically comprise fluorescent groups so that specific target/probe interactions can be read out using fluorescence optics with high spatial resolution and at little expenditure compared with other conventional detection methods, particularly mass-sensitive methods (A. Marshall, J. Hodgson, DNA chips: An array of possibilities, Nature Biotechnology 1998, 16, 27-31; G. Ramsay, DNA Chips: State of the art, Nature Biotechnology 1998, 16, 40-44).
Depending on the substance library immobilised on the micro-array and the chemical nature of the target molecules, interactions between nucleic acids and nucleic acids, between proteins and proteins as well as between nucleic acids and proteins can be investigated using this test principle (for a review see F. Lottspeich, H. Zorbas, 1998, Bioanalytik, Spektrum Akademischer Verlag, Heidelberg Berlin).
Antibody libraries, receptor libraries, peptide libraries and nucleic acid libraries can be considered as substance libraries that can be immobilised on micro-arrays or chips.
The nucleic acid libraries play by far the most important role. These are micro-arrays on which deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules are immobilised.
A prerequisite for the binding of a target molecule in the form of a DNA or RNA molecule and labelled with a fluorescence group to a nucleic acid probe of the micro-array is that both the target molecule and also the probe molecule are present in the form of a single-stranded nucleic acid. Efficient and specific hybridisation can only take place between such molecules. Single-stranded nucleic acid target molecules and nucleic acid probe molecules are generally obtained by heat denaturing and optimal choice of parameters such as temperature, ionic strength and concentration of helix-destabilising molecules. It is thus ensured that only probes with almost perfectly complementary sequences, i.e., corresponding to one another, remain paired with the target sequence (A. A. Leitch, T. Schwarzacher, D. Jackson, I. J. Leitch, 1994, In vitro Hybridisierung, Spektrum Akademischer Verlag, Heidelberg Berlin Oxford).
A typical example for the use of micro-arrays in biological test methods is the detection of micro-organisms in samples in biomedical diagnostics. In this case, use is made of the fact that the genes for ribosomal RNA (rRNA) are ubiquitously distributed and have sequence sections which are characteristic for the particular species. These species-specific sequences are deposited on a micro-array in the form of single-stranded DNA oligonucleotides. The target DNA molecules to be analysed are first of all isolated from the sample to be analysed and are provided with markers, for example fluorescent labels. The thus labelled target DNA molecules are then incubated in a solution with the probes deposited on the micro-array, non-specifically occurring interactions are removed by suitable washing steps and specific interactions are detected by fluorescence optical evaluation. In this way it is possible to detect for example a plurality of micro-organisms simultaneously in one sample. In this test method the number of detectable micro-organisms theoretically only depends on the number of specific probes which have been deposited on the micro-array.
For the practical implementation of these tests the micro-arrays or chips are fixed in closed chambers having inlets and outlets for changing liquids required for the washing and hybridisation steps. Such systems are described for example in U.S. Pat. No. 6,287,850 and WO 01/02094. DE 199 40 750 describes a support for analyte determining methods which following slight design modifications is suitable for use in array applications within the context of this invention.
Surface-bound DNA libraries which are deposited on slides are usually used for DNA sequence analysis. So far, special hybridisation chambers or incubation chambers have been used for carrying out the hybridisation reaction on these slides. In order to ensuring the tempering and mixing of the hybridisation solution in these hitherto known chambers, equipment specially adapted for the device used, which is therefore expensive and costly, is required.
DE 101 49 684.2 describes a flow cell which is suitable for carrying out a PCR as well as hybridisation reactions on DNA chips. The flow cell described therein is a complex structural element which is provided with a number of technical features which preclude the use of equipment conventionally used in laboratories such as a thermomixer for example (Eppendorf, Germany, Hamburg) or a laboratory centrifuge (Heraeus, Hanau, Germany).
WO 01/02094 describes a cartridge which comprises a DNA chip. In this cartridge both a PCR and a hybridisation reaction can be carried out on a DNA chip. WO 95/33846 describes a body with a recess in which a substrate with nucleic acid molecules of known sequence is deposited on predetermined regions. The body has a sealed cavity into which sample liquid can be injected. The filling channels are sealed up using septa and are opened with injection cannulae to fill the body or the cartridge. The use of the cartridges described hereinbefore likewise requires apparatus specifically provided for this purpose.
U.S. Pat. No. 5,856,174 describes a miniaturised integrated nucleic acid diagnostics device. This device can be used for the collection of one or a plurality of samples, their preparation and the subsequent execution of a plurality of sample analyses. Such a device can be used for automatically carrying out a DNA-chip-based analysis by combining and miniaturising all the steps incurred on a cartridge. The provision of such a device is extremely expensive and costly.
U.S. Pat. No. 5,545,531 describes a method for manufacturing microtitre plates whose bottom is a wafer which has a sample matrix at each position at which a recess is located in the microtitre plate. U.S. Pat. No. 5,874,219 describes a method for the concurrent performance of biological tests in which a plurality of reaction vessels are arranged coherently next to one another and each vessel is provided with a molecular sample matrix. This biological chip reaction vessel plate is configured such that the interactions on the molecular sample matrix can be read out with suitable readers. In this way, a number of biological samples can be investigated next to one another and parallel with molecular sample matrices. The reaction vessel plate described there is not suitable for carrying out individual tests. The manufacture of such a reaction vessel plate is described in U.S. Pat. No. 5,545,531.
In the light of the prior art described hereinbefore it is obvious that there is a great need for apparatus which on the one hand can be provided simply and cost-effective and on the other hand, can be used for carrying out micro-array-based detection tests simply. In particular, there is a need for devices for carrying out micro-array-based tests which allows the use of typical equipment and instruments in everyday use in the laboratory. In general, there is a need for equipment for carrying out micro-array-based tests which is distinguished by a simple design, easy handling, avoidance of contamination sources, carrying out reproducible tests and low manufacturing costs.
At the present time, analyses based on probe arrays are thus generally read out using fluorescence optics (see A. Marshall and J. Hodgson, DNA Chips: An array of possibilities, Nature Biotechnology, 16, 1998, 27-31; G. Ramsay, DNA Chips: State of the Art, Nature Biotechnology, 16, January 1998, 40-44). A disadvantage with the conventional detection methods, however, is the considerable technical effort in some cases and the high costs associated with the detection method.
Recently a number of array methods have been developed which allow qualitative and/or quantitative detection of the interaction between probes and targets with relatively low technical effort.
DE 100 33 334.6 and WO 02/02810 describe methods for the quantitative and qualitative detection of molecular interactions on probe arrays by time-resolved precipitation reactions as well as the relevant equipment and single-use articles.
WO 99/35499 describes a device in which substance libraries are addressably deposited on a disk which is similar to a modern compact disk (CD). A certain substance on the disk can be approached by rotating the disk. The read head of the reader can then be moved along the radius of the disk. The position of the disk can be determined by tracks which are integrated and which can be read out with a standard CD player. The interaction of the sample with a target substance can be accomplished by absorption measurements and fluorescence measurements in transmitted or incident light. Equally, magnetic particles which can be detected using a magnetic read head can be deposited. In order to visualise the interaction reaction, staining by a silver precipitate is especially proposed, whose precipitation is mediated by a streptavidin/biotin-gold conjugate.
WO 00/72018 discloses a method for the precipitation of silver on slides for visualising interaction reactions of substance libraries. The readers required for this purpose are also described.